PROJECT SUMMARY mRNA export is a fundamental component of the gene expression program in eukaryotes, which is tightly linked to upstream (e.g. transcription) and downstream (e.g. RNA surveillance and translation) events. Development of mRNA export models and an understanding of the role export plays in regulated gene expression (e.g. biased export of select transcripts) requires a quantitative assessment of mRNA export that encompasses kinetics and molecular mechanism. Pursuing this goal is timely given the knowledge that has amassed and technology developed (e.g. RNA-aptamer tagging systems) and is vital to understanding how disease arises in the context of perturbations to this cellular process. A key factor to consider is Dbp5p, a DEAD-box protein (DBP) that plays a critical role in export by modulating RNA-protein interactions important to directional transport of mRNAs through nuclear pore complexes (NPCs). Dbp5p is activated (i.e. enhanced ATPase activity) by the NPC components Gle1p and Nup159p, and although the impact of these regulators have been studied in vitro (e.g. steady-state kinetics and structural biology), the kinetic and thermodynamic origins of this regulation, and how ATPase cycling influences spatial and temporal aspects of mRNA export in vivo, remain unresolved. Therefore, a necessary step towards accurate models of mRNA export are a quantitative understanding of (1) ATP utilization by Dbp5p and (2) the impact of Dbp5p on mRNA export dynamics in vivo, including how these events are coupled to interactions with NPC regulatory factors. Specifically, knowledge of the rate and equilibrium constants dictating Dbp5p ATPase activity and interactions with Nup159p and Gle1p is necessary for developing predictive NPC transport models that can be combined with measurements of mRNA export kinetics and efficiency in vivo. Proposed research efforts in Aim 1 focus on transient (pre-steady state) kinetic and quantitative equilibrium methods and analyses to determine how Nup159p and Gle1p interact with and regulate Dbp5p. Aims 2 and 3 employ in vivo studies using single particle RNA imaging and transcriptomic approaches to address spatial and temporal aspects of mRNA export, including the role of Dbp5p, Gle1p and Nup159p in modulating export. To accomplish these aims, new experimental and analysis methods readily applicable to other DBPs and RNA transport systems will be developed. Completion of the proposed activities will establish a quantitative molecular framework that describes how regulatory factors function to modulate Dbp5p ATPase activity and mRNA transport, which is fundamental to gene expression, cell physiology, and disease pathology. !